Oliver Xiaoou Dong, Shu Yu, Rashmi Jain, Nan Zhang, Phat Q. Duong, Corinne Butler, Yan Li, Anna Lipzen, Joel A. Martin, Kerrie W. Barry, Jeremy Schmutz, Li Tian & Pamela C. Ronald 

Nature Communications volume 11, Article number: 1178 (2020) Cite this article

Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.

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